体外研究 研究结膜杯状细胞中基于性别的差异的方法
Summary
在基于性别的差异研究中，无酚红/无胎牛血清培养基是比晚期 RPMI 更好的选择，可在不改变结膜杯状细胞正常功能的情况下消除外源激素。
Abstract
干眼症是一种影响眼表健康的多因素疾病，女性的患病率要高得多。结膜杯状细胞 （CGC） 分泌到眼表的凝胶形成粘蛋白被破坏会导致多种眼表疾病。在CGC基于性别的差异的体外研究期间，消除外源性激素对于获得一致的结果至关重要。本文描述了一种在CGCs基于性别的差异研究中最大限度地减少外源性激素存在的方法，同时保持其生理功能。将来自死后人类供体的CGC在含有10%胎牛血清（FBS）（称为完全培养基）的RPMI培养基中从结膜碎片中培养，直至汇合。在实验开始前近48小时，将CGC转移到不含酚红或FBS但含有1%BSA（称为无酚红培养基）的RPMI培养基中。通过使用呋喃 2/乙酰氧基甲基 （AM） 显微镜测量卡巴酚 （Cch， 1 x 10-4 M） 刺激后细胞内 [Ca 2+] （[Ca2+]i） 的增加来研究正常细胞功能。结果表明，CGCs在无酚红培养基中48 h后仍能维持正常功能。在Cch刺激下，无酚红RPMI培养基和完全培养基之间的[Ca2+]i反应没有显著差异。因此，在基于性别的差异研究中，我们推荐使用含有 1% BSA 的不含酚红的 RPMI 培养基来消除外源激素，而不会改变 CGC 的正常功能。
Introduction
基于性别的差异影响眼表的多个过程 1,2,3。这些基于性别的差异的临床表现是男性和女性之间许多眼表疾病患病率的差异，例如干眼症和结膜炎4,5,6。有证据表明，基于性别的差异源于多个生物学水平，包括 X 和 Y 染色体7 上基因的不同特征以及激素的影响8。研究基于性别的差异的分子基础可以更好地了解疾病，并最终改善个性化医疗。
眼表包括覆盖的泪膜、角膜和结膜。在眼表的多个组成部分中观察到基于性别的差异，包括泪膜 9,10、角膜 11、泪腺12,13 和也分泌泪液的睑板腺12。许多机制研究调查了性激素对角膜及其相关成分的影响14,15;然而，人们对结膜及其杯状细胞的性别差异知之甚少。结膜是覆盖硬膜和眼睑内表面的粘膜。结膜上皮由非角化、多层、分层的鳞状细胞组成16。
在结膜的分层鳞状细胞中，有散布在上皮顶端表面的杯状细胞（CGC）。这些杯状细胞的特征在于位于顶极17的大量分泌颗粒。CGCs合成并分泌凝胶形成粘蛋白MUC5AC，以滋润眼表并在眨眼时润滑眼表17。粘蛋白分泌受到细胞内 [Ca 2+] （[Ca2+]i） 和 Ras 依赖性细胞外信号调节激酶 （ERK1/2）18 的激活的严格调节。不能分泌粘蛋白会导致眼表干燥和病理异常的后遗症。然而，在发炎的眼表上，炎症介质刺激的大量粘蛋白分泌导致眼睛的粘稠感和瘙痒感19.这些粘蛋白分泌紊乱的情况最终会导致眼表恶化。
杯状细胞作为眼粘蛋白主要来源的作用早已得到认可20，然而，生理和病理状态下粘蛋白调节的性别差异仍未被发现。体外系统可用于监测杯状细胞的功能，而没有激素效应或精确控制性激素水平。尽管结膜上皮细胞系已经发育了21 种，但没有具有功能性粘蛋白分泌的杯状细胞系可用。因此，我们修改了我们开发的原代人类CGC培养物，以建立一种分析体外性别差异的方法16，并呈现如下。
Protocol
所有人体组织均经捐献者事先知情同意和授权捐献至眼库，用于科学研究。马萨诸塞州眼耳人类研究委员会审查了人类结膜组织的使用，并确定为豁免，不符合人类受试者研究的定义。 1. 原代人杯状细胞培养 从眼库中，获得人结膜组织16. 制备培养基，补充有10%胎牛血清（FBS），2mM谷氨酰胺，2mM非必需氨基酸（NEAA），2mM丙酮酸钠?…
Representative Results
原代培养物中的人 CGC 在大约 14 天内增长到 80% 汇合度。通过对杯状细胞标志物CK7和HPA-125 的抗体进行免疫荧光染色来确认细胞类型（图1）。尽管从培养基中去除FBS可以消除性激素，但缺乏FBS可能会影响细胞反应。为了验证激素消除方法，使用胆碱能激动剂（卡巴胆碱，Cch 1 × 10-4 M）作为刺激物，以模拟由杯状细胞周围的不同神经末梢介导的生理性…
Discussion
研究眼部组织中基于性别的差异有助于了解疾病的过程，尤其是干眼症和过敏性结膜炎，它们不成比例地影响一种性别 4,5,6。尽管动物模型可用于这些研究，但由于与体内人类细胞具有最高的相似性，直接从人体组织获得的数据是必不可少的。当前实验环境中使用的结膜组织来自年龄范围为 70-85 岁（绝经后）的已故供?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作由美国国家眼科研究所资助EY019470（DAD）资助。
Materials
| 0.05% trypsin with 1x EDTA | Gibco (Grand Island, NY) | 25300-054 | |
| 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid | Fisher Bioreagent (Pittsburgh, PA) | BP310-500 | |
| Advanced RPMI media | Gibco (Grand Island, NY) | 12633020 | |
| carbachol | Cayman Chemical (Ann Arbor, MI) | 144.86 | |
| Fetal Bovin Serum | R&D (Minneapolis, MN) | S11150H | |
| Fura-2- acetoxymethyl ester | Thermo Fisher Scientific (Waltham, MA, USA) | F1221 | |
| Human conjunctival tissue | Eversight Eye Bank (Ann Arbor, MI) | N/A | |
| inorganic salt for KRB buffer | Sigma-Aldrich (St. Louis, MO) | Any brand will work | |
| L-glutamine | Lonza Group (Basel, Switzerland) | 17-605F | |
| non-essential amino acids | Gibco (Grand Island, NY) | 11140-050 | |
| penicillin/streptomycin | Gibco (Grand Island, NY) | 15140-122 | |
| phenol red-free RPMI media | Gibco (Grand Island, NY) | 11835055 | |
| Pluronic acid F127 | MilliporeSigma (Burlington, MA, USA) | P2443-250G | |
| RPMI-1640 culture medium | Gibco (Grand Island, NY) | 21875034 | |
| scalpel | Thermo Fisher Scientific (Waltham, MA, USA) | 12460451 | Any sterile surgical scalpel can work |
| sodium pyruvate | Gibco (Grand Island, NY) | 11360-070 | |
| sulfinpyrazone | MilliporeSigma (Burlington, MA, USA) | S9509-5G |
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Citer Cet Article
Bair, J. A., Dartt, D. A., Yang, M. In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells. J. Vis. Exp. (197), e64456, doi:10.3791/64456 (2023).